Linear amplification of mRNA or genomic DNA using in vitro transcription, or IVT, is a well-known method of molecular biology (see Krieg & Melton, 1984, Melton, 1984). Because IVT, for each target, produces a number of RNA products that is proportional to the original number of copies of that target, it permits the determination of relative message abundance (U.S. Pat. Nos. 5,545,522; 5,716,785; and 5,891,636; Van Gelder et al, 1990) and thus has been widely applied in the context of gene expression analysis (U.S. Pat. No. 5,514,545). IVT also is a central element in certain isothermal methods of exponential target amplification which are capable of detecting pathogen RNA or mRNA at low levels (U.S. Pat. No. 5,399,491; European Patent No. 0 368 906 B2; Guatelli et al., 1990).
In accordance with prior art, as disclosed in U.S. Pat. Nos. 6,291,170 and 5,514,545, as well as in U.S. Patent Applications 2005/0130194 and 2005/0123943, the conventional sequence of step is as follows: cDNA synthesis is performed, most frequently using a primer complementary to polyA 3′-end of RNA which includes a T7 promoter sequence (non-template strand) at its 5′-end; alternatively, sequence or gene-specific primers may be placed in positions other than the 5′-end. After RNaseH digestion of the RNA template or heat denaturation of RNA-DNA hybrid, second strand DNA synthesis is performed (Goubler, U., 1983), to produce dsDNA of full or partial length (depending on primer placement), incorporating a double-stranded T7 promoter sequence (and adjacent regions). In practical realizations of the method, DNA polymerase or RT must be added to the reaction to effectively catalyze second strand synthesis (see U.S. Pat. No. 5,545,522, describing use of E. Coli DNA Polymerase; Kwoh et al., 1989). Antisense RNA (aRNA) is synthesized from the second strand of DNA by in-vitro transcription, and the aRNA products are detected, for example by hybridization to capture oligonucleotide probes, including variants such as molecular beacons (Vet, J. A. M., 2002; see also BioArray Solutions patent application Ser. No. 11/218,838; filed Sep. 2, 2005, below) or the hybridization protection assay (see U.S. Pat. No. 6,004,745; Arnold et al.), or probe elongation. All these methods of the art require the synthesis of double-stranded cDNA from the original mRNA targets, and the intervening step of RNA degradation. The complex and time-consuming steps of these methods have effectively confined them to the laboratory research. In a clinical setting, the use of such complex protocols would require special training, and often certification, of technical staff in laboratories qualified to conduct such complex (“esoteric”) analysis.
Nucleic Acid Detection and Sequence Analysis—IVT also can be applied to DNA analysis, including mutation or polymorphism analysis. Generally, these applications require exponential amplification of genetic material i.e., genomic DNA, most commonly by application of the polymerase chain reaction (Syvanen, A. C., 2005, see, e.g., U.S. Pat. No. 4,683,202; Mullis) or whole genome amplification, in a multiplicity of variants (see, e.g. USCD patents on ligation-mediated whole genome amplification U.S. Pat. No. 5,686,243). IVT offers a method of strand selection following PCR amplification (see, e.g., BioArray Solutions Application filed Sep. 2, 2005; Ser. No. 11/218,838; filed (IVT)) which, inter alia, has the advantage of permitting the combination of that step with subsequent multiplexed detection of RNA strands produced in the IVT reaction.
It will be useful to simplify and accelerate the design of reliable multiplexed amplification and detection reactions, and to replace the complex procedures for gene expression analysis (U.S. Pat. No. 5,514,545) and other tasks of nucleic acid analysis by simpler, more robust protocols suitable for the clinical setting. Especially in that context, it also will be useful to develop integrated protocols, that is, protocols which combine multiple steps of analysis, preferably in a manner permitting the realization of homogeneous assay formats. It will be especially useful, in order to reduce the time required for assay completion, and particularly “hands-on” time, to combine amplification and analysis, by detection of multiple amplification products. Further, the combination of steps, preferably in a manner compatible with the realization of homogeneous assay formats, will facilitate miniaturization, which in turn will to reduce the consumption of reagents as well as the risk of contamination, both of samples and of laboratory facilities.
An IVT reaction—and in particular, an IVT reaction using a single-stranded template (rather than a double-stranded template), as described herein—offers many of these advantages.
In fact, the ability of the T7 RNA polymerase to utilize the template strand of the promoter in single-stranded form, and catalyze transcription from a single-stranded template (sst) producing a copy of the parent DNA strand of interest, has been described in the literature (Kukarin, A. et al, 2003; Korencic D. et al, 2002; Temiakov D. et al, 2002). However, in practical implementations of (conventional) IVT, this reaction has been regarded as an adverse side effect of in-vitro transcription.
The sst-IVT reaction, to date, has not been fully applied to the development and realization of complex analytical protocols, primarily because of certain generally undesirable characteristics. First, its modest yield—compared to the regular IVT format using double-stranded (ds) DNA templates—limits the sensitivity of assay protocols performed in conventional configurations that require a substantial number of target molecule; and its poor performance in buffers of even modest ionic strength generally renders it incompatible with other upstream and downstream enzymatic reactions employed in existing assay protocols. However, by addressing these points as described below, sst-IVT can be optimized to make it suitable for numerous applications of nucleic acid analysis, in a manner permitting the integration of amplification and concurrent detection and analysis of multiple products.